Downregulation of Kinesin Spindle Protein Inhibits Proliferation, Induces Apoptosis and Increases Chemosensitivity in Hepatocellular Carcinoma Cells

Background: Kinesin spindle protein (KSP) plays a critical role in mitosis. Inhibition of KSP function leads to cell cycle arrest at mitosis and ultimately to cell death. The aim of this study was to suppress KSP expression by specific small-interfering RNA (siRNA) in Hep3B cells and evaluate its anti-tumor activity. Methods: Three siRNA targeting KSP (KSP-siRNA #1-3) and one mismatched-siRNA (Cont-siRNA) were transfected into cells. Subsequently, KSP mRNA and protein levels, cell proliferation, and apoptosis were examined in both Hep3B cells and THLE-3 cells. In addition, the chemosensitivity of KSP-siRNA-treated Hep3B cells with doxorubicin was also investigated using cell proliferation and clonogenic survival assays. Results: The expression of endogenous KSP at both mRNA and protein levels in Hep3B cells was higher than in THLE-3 cells. In Hep3B cells, KSP-siRNA #2 showed a further downregulation of KSP as compared to KSP-siRNA #1 or KSP-siRNA #3. It also exhibited greater suppression of cell proliferation and induction of apoptosis than KSP-siRNA #1 or KSP-siRNA #3; this could be explained by the significant downregulation of cyclin D1, Bcl-2, and survivin. In contrast, KSP-siRNAs had no or lower effects on KSP expression, cell proliferation and apoptosis in THLE-3 cells. We also noticed that KSP-siRNA transfection could increase chemosensitivity to doxorubicin in Hep3B cells, even at low doses compared to control. Conclusion: Reducing the expression level of KSP, combined with drug treatment, yields promising results for eradicating hepatocellular carcinoma (HCC) cells in vitro. This study opens a new direction for liver cancer treatment.

INTRODUCTION epatocellular carcinoma (HCC) is the fifth common cancer in the world. Due to the lack of an early clinical diagnosis method and obvious symptoms, HCC tumor is regularly detected at advanced stages. Although surgical operation on tumor removal is generally helpful for HCC patients, clinical mortality of HCC is relatively high because therapeutic options of HCC are rather limited [1]. Besides, traditional chemotherapies using anti-cancer drugs or radiotherapy are inefficacious for HCC patients. In many cases, this combined therapy still leaves some problems such as metastatic lesions and subsequent cancer recurrence [2]. Recently, following the development of modern molecular biology, in-depth researches have been conducted in developing new strategies of HCC treatment at genetic level. In particular, RNA interference (RNAi) may represent a powerful strategy to interfere with key molecular pathways involved in cancer and has established a new area of clinical therapy for HCC [3].
RNAi is a process in which activation of an intracellular pathway modulated by small-interfering RNA (siRNA) composed of 21-23 nucleotides (nt) leads to degradation of a specific, targeted mRNA [4]. The selective and robust effect of RNAi on gene expression makes it a valuable research tool both in cell culture and in living organisms because synthetic siRNA introduced into cells can induce suppression of specific genes of interest [5]. Another unique advantage of RNAi is that non-druggable protein H targets can be efficiently knocked-down and possibly achieve therapeutic effects [6]. Therefore, siRNAinduced RNAi presents an effective and a simple method to silence a wide range of cancer-associated genes. Moreover, a number of siRNA have been established that are capable of silencing some different types of human HCC gene targets, such as cyclin E, vascular endothelial growth factor, COP9 signalosome subunit 5, c-Myc [7][8][9][10] and so on.
It has been known that human cancer is a generelated disease involving abnormal cell growth. As a new member of the kinesin superfamily of microtubule -based motors, the kinesin Eg5, also called kinesin spindle protein (KSP) or KIF11 is a molecular motor that participates in mitosis, by separating the microtubules that are attached to two centrosomes, thus contributing to the bipolar arrangement of the spindle [11]. Failure to establish a bipolar spindle results in a mitotic arrest, after which cells may experience a variety of fates, including abnormal exit from mitosis, resumption of the cell cycle, and apoptosis [6]. The overexpression of KSP as a transgene may cause genomic instability and tumor formation in mice [12,13]. Furthermore, in contrast to microtubules which are also presented in post-mitotic cells, KSP is exclusively expressed in mitotic cells, which make it an ideal target for anti-mitotics [14]. Inhibition of KSP activity leads to cell cycle arrest of mitotic cells in prometaphase with the formation of monoastral microtubule arrays and eventually to cell death [15,16]. Therefore, several KSP inhibitors have been studied in clinical trials and provided new opportunities for development of novel anti-cancer therapeutics alternative from the available microtubule targeting drugs [17][18][19].
In this study, siRNA targeting KSP was used to reduce KSP expression in Hep3B cells and to investigate the chemosensitivity of the KSP downregulated cells to doxorubicin. Our evidence indicates KSP-silencing efficiency and the impact of KSP downregulation combined with chemotherapy on the growth of Hep3B cells in vitro.
Transient transfection of siRNA. The sequences of the siRNA targeting KSP (KSP-siRNA#1-3) and mismatched siRNA (Cont-siRNA) are shown in Table  1. All siRNA were synthesized by Bioneer Co., Ltd (Daejeon, Republic of Korea). Each siRNA was resuspended in nuclease-free water, and the stock solutions were stored at 4 o C until use. The KSP-siRNA#1-3 or Cont-siRNA was transiently transfected in both Hep3B cells and THLE-3 cells with a Lipofectamine RNAiMAX Transfection Reagent kit (Invitrogen Inc., Carlsbad, CA, USA) by reverse transfection protocol. Briefly, for each well of 24-well plate (Corning Inc., NY, USA), 3 µl of siRNA duplex (20 µM) was mixed with 1 µl transfection reagent and 100 µl Opti-MEM medium supplied with hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, and growth factors. Then, the siRNAtransfection reagent complex was incubated with 500 µl of diluted cells (5 × 10 4 cells/well) at 37°C for 24-

Reverse transcription PCR (RT-PCR).
Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA). The concentration of RNA was measured using a BioPhotometer (Eppendorf, Hamburg, Germany). One-step RT-PCR was performed from total RNA using Access Quick RT-PCR kit (Promega, Madison, WI, USA) under the following conditions: initial reverse transcription at 45°C for 45 min and 95°C for 2 min, followed by 35 cycles of denaturing at 94°C for 45 s, annealing at 55-58°C for 30 s, and extension at 72°C for 45 s. After completion of the last cycle, all samples were incubated at 72 o C for 10 min. The sequences of primers are shown in Table 2. PCR products were analyzed by electrophoresis with 2% agarose gel, visualized with EtBr staining (Sigma-Aldrich, St. Louis, MO, USA) and photographed by Bioimaging system (GELDOC-IT, UVP, Upland, CA, USA).

Real-time quantitative reverse transcription PCR (real-time qRT-PCR).
Real-time qRT-PCR was carried out with SYBR Green One-Step qRT-PCR kit (Invitrogen Inc., Carlsbad, CA, USA) under the following conditions: initial reverse transcription at 45°C for 30 min and 95°C for 3 min, followed by 35 cycles of denaturing at 94°C for 45 s, annealing at 55-58°C for 30 s, and extension at 72°C for 45 s. Internal calibration curves were generated by the real-time software (version 2.2). A melting curve analysis was carried out between 60°C and 95°C with a plate read every 0.5°C after holding the temperature for 20 s. The cycle number (Ct) at which the signals crossed a threshold set within the logarithmic phase and the peaks of melting curves were recorded. The relative quantitation of gene expression in terms of fold change was calculated using the 2 -ΔΔCt method [20]. Relative expression levels of target genes in each treatment group were derived from normalizing the Ct value of target genes against that of an endogenous reference (β-actin) and a calibrator (control cells).
Apoptosis assay. Apoptosis was investigated by flow cytometry using Annexin V and propidium iodide (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, the cell concentration was firstly adjusted to 1 × 10 6 cells/ml, and then 1 ml of the cell suspension was taken and centrifuged at 500 ×g at 4 o C for 10 min. The pellet was rinsed twice with PBS and then re-suspended in a proper volume of binding buffer so that the cell concentration was 5 × 10 4 cells/ml. After the addition of 10 µl Annexin V-fluorescein isothiocyanate and 5 µl propidium iodide followed by gentle mix, a 15-min reaction was initiated in darkness at room temperature. After that, 300 µl binding buffer was added, and flow cytometry was performed using CellQuest Pro software (BD Biosciences, Franklin Lakes, NJ, USA) to detect cell apoptosis rate (%).

Anti-tumor drug treatment assay.
To investigate whether the transfection of KSP-siRNA increases chemosensitivity of Hep3B cells, KSP-siRNA-treated cells were plated at a density of 3 × 10 3 cells per well in 96-well plates (Corning Inc., NY, USA) and incubated with doxorubicin (Sigma-Aldrich, St. Louis, MO, USA) at various concentrations: 0, 1, 2, and 4 μg/ml for 24 h. Cont-siRNA-treated cells and untreated control cells were also grown under the same conditions. Using WST-1 assay and clonogenic survival assay, cell proliferation was analyzed for indicated time after treatment.
Clonogenic survival assay. The clonogenic survival assay was used to determine the capacity for cell survival and proliferation after radiation or chemotherapy [21]. After treatment with siRNA, cells were seeded at a density of 100 cells per well in 6-well plates for 24 h in a complete medium followed by treatment with different concentrations of doxorubicin: 0, 1, 2, and 4 μg/ml. After 24 h, the medium was replaced with a fresh medium and incubated for additional 10 days. Clones were fixed with methanol and stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for about 15 min. Stained clones that had more than 50 cells were counted at low magnification and cloning efficiency calculated as follows: Cloning efficiency = (Clone number/Total cell number) × 100%.

Statistical analysis.
Each experiment was performed in triplicate for all data (n = 3). Data are expressed as mean ± standard error of the mean. Statistical comparisons were performed using the Student's t-test and ANOVA. P values < 0.05 were considered to be statistically significant.

The expression of endogenous KSP in Hep3B cells and THLE-3 cells.
In this study, the expression of endogenous KSP at mRNA and protein levels were determined in both HCC cell line Hep3B, and normal liver cell line THLE-3 by RT-PCR and Western-blot analyses. Density of KSP band in RT-PCR analyses showed that endogenous KSP expression at mRNA level was higher in Hep3B cells compared to THLE-3 cells (control cells in Fig. 1a and c). It was also confirmed at protein level by Western-blot analyses (control cells in Fig. 2a and c).  (Table 1). Then, one siRNA with best repressive effect was used in following experiments.

Effects of KSP-siRNA on KSP expression in Hep3B cells and THLE-3 cells. To address the functions of KSP, both Hep3B cells and THLE
In THLE-3 cells, KSP-siRNA had less influence on KSP expression at both mRNA and protein levels. However, there was no significant difference in the expression of KSP between KSP-siRNA-treated cells or Cont-siRNA-treated cells and untreated control ones ( Fig. 1c and d, Fig. 2c and d). In contrast, in examined Hep3B cells, all three KSP-siRNA had noticeable effects on KSP expression. Among them, KSP-siRNA#2 was the most effective in reducing KSP-mRNA, and the downregulation was correlated with a remarkable decrease in target protein ( Fig. 1a and b, Fig. 2a and b). Therefore, KSP-siRNA#2 was selected to evaluate its effects on KSP expression in Hep3B cells at different time intervals after transfection. The results of RT-PCR experiments on total RNA, obtained from the KSP-siRNA#2-Hep3B cells, indicated downregulation of KSP-mRNA in comparison to that of control cells and Cont-siRNA-treated cells after 72 h. The density of KSP band showed that KSP-mRNA expression was blocked clearly after 48 h, and inhibition was stabled up to 72 h after transfection (Fig.  3a). In addition, the relative amounts of KSP-mRNA in each sample were quantified using real-time qRT-PCR.
The results of real-time qRT-PCR analyses indicated that mRNA expression level of KSP in KSP transfected cells began to alter within transfection, from 100% to 77.85 ± 3.15% expression level was strongly decreased after   (Fig. 3b). These values indicated mRNA expression in Hep3B cells and THLE-3 cells. Cells were transfected with KSP and Cont-siRNA. Total RNA was extracted from cells at 72 h after siRNA transfection products of the KSP (480 bp) and β-actin (680 bp) genes were exhibited in Hep d) Quantitative analyses of KSP-mRNA level were determined by real-time qRT cells (d). The mRNA expression of KSP was normalized with β SD) of triplicate. * P<0.05 and ** P<0.01 compared to control cell group. Chemosensitivity 5 h (20.47 ± 2.69%) as control cells (p < 0.01), while it siRNA-transfected cells after transfection (93.35 ± 3.85%) indicated that KSP-siRNA#2

Downregulation Inhibits Proliferation, Induces Apoptosis and Increases
Cells were transfected with KSPh after siRNA transfection. (a genes were exhibited in Hep3B cells (a) and time qRT-PCR after siRNA KSP was normalized with β-actin. Each bar represents control cell group.  siRNA might correlate with the extent of endogenous KSP expression which was higher in compared to non-cancerous

Effects of KSP-siRNA on cell proliferation Hep3B cells and THLE-3 cells.
The effects of on the viability of both Hep3B cells and were determined at 72 h after transfection cell viability was examined by WST liferation assays. In Hep3B cells, all three KSP induced a significant reduction in cell compared to untreated control cells (P<0.05 The results clearly showed that cell proliferation was markedly inhibited by KSP-siRNA. Consistent with the changes in KSP expression, KSP-siRNA#2 the greatest suppression on cell growth, thus selecting for further studies. Compared to Hep3B siRNA displayed no or lower effects on the viability of THLE-3 cells (Fig. 4b). In addition, we did not observe any significant phenotypic differences when KSP siRNA was used in THLE-3 cells (data not shown both Hep3B cells and THLE-3 cells, transfection with Cont-siRNA did not affect cell viability hand, the negative Cont-siRNA had relatively limited off-target effects on cell proliferation These results displayed that cell growth inhibition conformable with the extent of KSP siRNA in both Hep3B cells and THLE-3 siRNA on cell proliferation in effects of siRNA cells and THLE-3 cells after transfection (Fig. 4). The was examined by WST-1 cell proall three KSP-siRNA on in cell viability 0.05) (Fig. 4a). results clearly showed that cell proliferation was Consistent with the #2 also caused , thus selecting B cells, KSPlower effects on the viability of , we did not observe phenotypic differences when KSPdata not shown). In transfection with viability. On the other siRNA had relatively limited cell proliferation as expected.
that cell growth inhibition was silencing by cells.

Effects of KSP-siRNA#2 on apoptosis cells and THLE-3 cells.
The growth inhibition in downregulated Hep3B cells could be attributed in part to cell apoptosis. The apoptosis was assessed by Annexin-V staining methods. In the staining method, the percentage of apoptotic cells was scored by using flow cytometry. Our results apoptosis in Hep3B cells after treatment with KSP siRNA#2 (Fig. 5a). The percentage of apoptotic cells by KSP-siRNA#2 treatment was exponentially changed after 24 h (8.86 ± 0.78 increased after 48 h (12.25 ± 1.42% 0.65%), respectively as compared cells

on apoptosis in Hep3B
The growth inhibition in KSPcells could be attributed in part The apoptosis was assessed by V staining methods. In the staining method, apoptotic cells was scored by using flow cytometry. Our results revealed induction after treatment with KSPpercentage of apoptotic cells treatment was exponentially 0.78%) and significantly 1.42%) and 72 h (20.64 ± compared to that of control , whereas the apoptosis treated cells at 72 h was Fig. 5b). Consequently, B cells transfected with was higher than control cells or Contver, the result was not cells, which showed no apoptosis rate between KSPtreated cells and control cells KSP silencing (Fig. 5c and d). It that siRNA-mediated down-B cells could induce of proliferation.

The molecular mechanisms underlying the growth inhibitory effects and apoptosis induction of KSP downregulation in Hep3B cells.
To identify the molecular targets and common mechanisms the growth inhibitory effects and apoptosis induction caused by KSP silencing, Hep3B cells were treated with either Cont-siRNA or KSP-siRNA# subjected to analysis anti-apoptosis gene expression, including cyclin D1, Bcl-2, and survivin indicated that downstream targets of cyclin D and survivin were also downregulated at and mRNA levels. The band intensity products showed the expression levels D1, Bcl-2, and survivin mRNA were lower of control cells and Cont-siRNA-treated cel

The molecular mechanisms underlying the growth inhibitory effects and apoptosis induction of KSP
To identify the molecular targets and common mechanisms underlying and apoptosis induction cells were treated #2 for 72 h and apoptosis gene expression, urvivin. The results yclin D1, Bcl-2, were also downregulated at both protein y of RT-PCR expression levels of cyclin lower than those treated cells, after 72 h (Fig. 6a). The relative levels of mRNA of D1, Bcl-2, and survivin were also determined using real-time RT-qPCR after 72 h of The mRNA levels of cyclin D downregulated by 56.35 ± 2.25 respectively, whereas the mRNA levels were downregulated by 51.34 siRNA#2-transfected cells compared cells (P<0.05) (Fig. 6b). Similarly, levels of cyclin D1, Bcl-2, and measured using Western-blot fection, indicating similar result siRNA#2 inhibited cyclin D1 expression at the protein level up to 54.35 ± 4.32%, and 51.56 ± 3.78%

Downregulation Inhibits Proliferation, Induces Apoptosis and Increases Chemosensitivity 9
and c) Cell apoptosis was the lower left (LL) quadrant represented survivals; lower -apoptotic and the upper left d) Data show the mean ± SD intensity of fluorescent positive cells during compared to control cell group.
levels of mRNA of cyclin were also determined using h of siRNA transfection. yclin D1 and Bcl-2 were 2.25% and 43.12 ± 3.02%, whereas the mRNA levels of survivin 51.34 ± 1.58% in KSPtransfected cells compared to those in control Similarly, the expression and survivin proteins were blot analyses after transresults (Fig. 7a). KSP-1, Bcl-2, and survivin protein level up to 46.55 ± 2.26%, 3.78%, respectively in comparison to control cells (P<0.05 However, there was no significant difference and protein levels of cyclin D1, Bcl-2, between Cont-siRNA-treated cells and untreated control cells. Electrophoretic profile of PCR 145 bp) and β-actin (680 bp) genes. β-actin was used as a housekeeping gene control and Survivin in Hep3B cells were also determined by real-time qRT expression of these genes was normalized with β-actin. Each bar represents the mean value ± standard deviation ( 0.05) (Fig. 7b). difference in mRNA , and survivin treated cells and untreated downregulated Hep3B cell oxorubicin. To on the viability , cells were treated for was determined using WST-1 KSP-siRNA#2 Hep3B cells in a -siRNA#2 had cells (Fig. 8a). cells, but not THLE-3 cells, were sensitive to addition, we also compared the cytotoxicity of C siRNA and KSP-siRNA#2 toward Hep days. Cells were treated with C siRNA#2 at same concentration that KSP-siRNA#2, but not Cont mediate cytotoxicity toward Hep To evaluate the inhibition e treatment on Hep3B cells, cells following treated doxorubicin at designated concentration indicated time were carried out in WST clonogenic assay. It was clear could significantly reduce Hep3 inhibitory effect exhibited in dependent manner (Fig. 9a). However doxorubicin on Hep3B cells were sensitive to KSP-siRNA. In addition, we also compared the cytotoxicity of Conttoward Hep3B cells for five ells were treated with Cont-siRNA or KSPat same concentration. The results showed ont-siRNA, can directly cytotoxicity toward Hep3B cells (Fig. 8b). To evaluate the inhibition effect of doxorubicin cells following treated with at designated concentrations for the were carried out in WST-1 assay and that doxorubicin alone 3B cell growth, and the in a dose-and time-However, the effects of were not observable at The size of each protein was indicated. (b) Densitometric analysis of these three proteins was made relative to represents the mean value ± standard deviation ( concentration of 1 μg/ml. At 4 μg/ml, the doxorubicin on cell proliferation became apparent with the inhibition rate value increasing 2.46% at day one to 51.71 ± 3.03% at addition, cloning efficiency was declined significantly in cells following treatment of 4 μg/ml dose when compared to control cells ( 10).  treated cells as well as Cont-siRNAtreated cells and control cells were treated with higher μg/ml) for five days. For ml or 4 μg/ml doxorubicin the inhibition rates were 80.64 ± 5.23% and For Cont-siRNA plus 2 doxorubicin groups, the inhibition 4.30% and 55.20 ± 4.16%, g/ml doxorubicin alone groups, the inhibition rates were 26.38 ± 54.46 ± 5.03%, respectively ( Fig. 9c and d addition, the KSP-downregulated cells showed no sign of proliferation, with necrosis observed at day after doxorubicin treatment ( Fig. 11 treatment with a series of doxorubicin presence of KSP-siRNA#2 increased the cell inhibition compared to treatment with doxorubicin and/or C siRNA, further supporting the synergistic effect other words, KSP-siRNA transfer can increase the doxorubicin chemosensitivity of Hep3B cell noted that the synergistic cytotoxic effect is even at low dose (1 μg/ml) compared to control results were also further supported by clonogenic survival assay. A significant decline efficiency was observed in the combination of siRNA#2 and doxorubicin as compared to siRNA#2 or doxorubicin alone (Fig. 10).

DISCUSSION
Several aspects of KSP or Eg5 biology excellent indicator for monitoring RNAi. First, the endogenous KSP gene is expressed in all proliferating cells that have been analyzed Second, its activity is required for mitosis, and therefore the penetration of siRNA systematically In downregulated cells showed no sign of proliferation, with necrosis observed at day three 11). Obviously, oxorubicin doses in the increased the cell inhibition oxorubicin and/or Cont-siRNA, further supporting the synergistic effect. In siRNA transfer can increase the B cell. It is also noted that the synergistic cytotoxic effect is effective, compared to control. These further supported by clonogenic assay. A significant decline in cloning the combination of KSPas compared to KSPbiology make it an RNAi. First, the KSP gene is expressed in all the analyzed to date. required for mitosis, and penetration of siRNA systematically results in an inhibition of growth. This inhibition can be evaluated by from cell counting to DAPI staining and either microscopy or cytofluorometry. Fin KSP-siRNA is rapid and best analyzed and 48 h. The evaluation of siRNA transfection in cells can therefore be easily monitored reagents required [16]. Therefore, degradation of KSP by siRNA was e a novel approach for the control of cancer cells decrease expression of KSP, we used RNAi technology to transfect siRNA targeting KSP into cancerous cell line Hep3B and non-cancerous cell line THLE control. Several siRNA targeting regions of human KSP were used routinely to ensure and possibly enhance silencing. transfection, cells were subjected to RT qRT-PCR, Western-blot, and drug effects were investigated. Transfection of KSP cells reduced the expression of KSP at protein levels. RT-PCR and analyses showed that KSP-mRNA KSP-siRNA-treated cells was much lower cells and Cont-siRNA-treated cells. level, KSP protein level of post cells were assessed by Western noticeable reduction in KSP expression observations are in consistent with that used KSP-siRNA to monitor KSP expression in different cancer cells, including lung carcinoma cells, breast carcinoma adenocarcinoma cells, and ovary cancer cells 22]. In contrast, in normal THLE expression of KSP in KSP-siRNA unaltered when compared to control cells. expression is elevated in tumor samples adjacent normal tissues in breast, colon, ovary, rectal, and uterus, consistent with its role proliferating cells [23]. Consequently, endogenous KSP expression may be a reason for decrease of KSP-silencing. This supported by a previous study correlation between the silencing efficiency of siRNA and the intracellular transcript levels different cell lines. The low abundant transcripts are less susceptible to siRNA-mediated degradation than the medium or high abundant trans Furthermore, gene silencing efficiency depend on specific features of the RNAi machinery in each cell lines [22] and the local structure of mRNA at the targeted region [25].
Inhibition of KSP activity, either by microinjection of antibodies [26] or with a monoastrol [15], leads to a monopolar spindle and an arrest of cells in prometaphase and ultimately death. In this work, we also demonstrated Proliferation, Induces Apoptosis and Increases Chemosensitivity 13 in an inhibition of growth. This growth inhibition can be evaluated by several approaches, DAPI staining and either cytofluorometry. Finally, the action of siRNA is rapid and best analyzed between 30 h of siRNA transfection in cells can therefore be easily monitored with no specific ]. Therefore, inducing a degradation of KSP by siRNA was expected to lead to a novel approach for the control of cancer cells. This data is also partially study which revealed a correlation between the silencing efficiency of siRNA and the intracellular transcript levels of target genes on low abundant transcripts are mediated degradation than medium or high abundant transcripts [24].
gene silencing efficiency of siRNA may depend on specific features of the RNAi machinery in and the local structure of mRNA at of KSP activity, either by microinjection specific drug such as leads to a monopolar spindle and an of cells in prometaphase and ultimately to cell demonstrated that KSP- siRNA#2 could inhibit cell proliferation apoptosis in cancerous Hep3B cells, but not cancerous THLE-3 cells. These results are in agreement with the previous observations when using several siRNA targeting KSP on both normal cells cancer cells [13,22]. The effects of KSP are also known to vary depending on cell line either inhibit cell proliferation or induce apoptosis To elucidate the molecular mechanisms KSP-siRNA inhibit proliferation and induce of Hep3B cells, we have examined the expressions of the key regulators cyclin D1, Bcl-2, and results first demonstrated that the expression levels of cyclin D1, Bcl-2, and survivin were significantly decreased in Hep3B cells after transfection with of KSP-siRNA. Cyclin D1 is known to accumulate during the G1 phase of the cell cycle [28]. Overexpression of cyclin D1 may be an early event in hepato carcinogenesis, and it plays a role in tumor differentiation [29,30]. In contrast, Bcl-2 are thought to be very important anti-apoptotic proteins in cells. They are identified to be mechanisms involved by cancer cells to evade apoptosis. Bcl-2 is the prominent member of a family that is responsible for dysregulation of apoptosis and prevention of death in cancer cells, which controls the pathways leading to the release of proliferation and induce but not in non-These results are in previous observations when using normal cells and effects of KSP knockdown known to vary depending on cell line and can or induce apoptosis [27]. molecular mechanisms by which induce apoptosis the expressions of and survivin. Our that the expression levels of urvivin were significantly transfection with of known to accumulate during . Overexpression of may be an early event in hepatoand it plays a role in tumor growth and 2 and survivin apoptotic proteins one of the mechanisms involved by cancer cells to evade is the prominent member of a protein family that is responsible for dysregulation of s and prevention of death in cancer cells, leading to the release of cytochrome c from the mitochondrial activation of caspase cascade and, execution of apoptosis [31]. 2 may protect human hepatoma cells from antibody mediated apoptosis [32]. Similarly, member of inhibitors of the apoptosis protein that have been implicated in both cell division and inhibition of apoptosis. By inhibiting apoptosis promoting mitosis, survivin facilitates cancer cell survival and growth. The overexpression of liver cancer can protect cells promote cell cycle progression apoptotic caspases-3 and caspases findings, we surmised that the downregulation of cyclin D1, Bcl-2, and survivin KSP transfection was one of inducing cell apoptosis, subsequently leading cell death. Drug resistance is a major problem in cancer treatment with chemotherapy, since after lon exposure to chemotherapeutic agent, cancer cells may no longer respond to the treatment. Or more too often, cancer cells, due to the intrinsic instability of their genome, may develop resistance to several completely different chemotherapeutic agents known as multidrug resistance [ of enhancing chemosensitivity by using apoptosis Iran. Biomed. J., January 2015 siRNA treated cells and KSP-siRNA#2 mL. Pictures were captured at cytochrome c from the mitochondrial membrane, the activation of caspase cascade and, in the end, to Overexpression of Bclmay protect human hepatoma cells from antibody-Similarly, survivin is a new inhibitors of the apoptosis protein family been implicated in both cell division and By inhibiting apoptosis and urvivin facilitates cancer cell overexpression of survivin in can protect cells from apoptosis and promote cell cycle progression by inhibiting procaspases-7 [33,34]. From our that the downregulation of expressions by siRNAthe important ways of , subsequently leading cell Drug resistance is a major problem in cancer chemotherapy, since after long term exposure to chemotherapeutic agent, cancer cells may no longer respond to the treatment. Or more too often, cancer cells, due to the intrinsic instability of their genome, may develop resistance to several completely different chemotherapeutic agents simultaneously, also [35]. Thus, the approach of enhancing chemosensitivity by using apoptosis-inducing agents appears to be a potential approach for more efficient cancer treatment. The inhibition of KSP expression by siRNA is anticipated to an increase in the chemosensitivity of cancer cells with anti-tumor drugs. Among anti-tumor drugs, doxorubicin is a commonly used anti-cancer drug causing DNA damage and killing cancer cells mainly by apoptosis. Doxorubicin is also used in many researches, especially on anti-tumor drug resistance. However, the process leading to death of cancer cells and molecular basis of resistance to doxorubicin are not well understood. In our study, Hep3B cells were treated with a series of doses of doxorubicin. The effects of doxorubicin on normal cells were not observable at low concentration. At higher doses, the effects on proliferation inhibition became apparent. It was suggested that Hep3B cells has strong resistance to doxorubicin. The resistance to doxorubicin was decreased by downregulation of KSP. In other words, the KSP knockdown cells were more sensitive significantly to doxorubicin compared to the original cells. Even at high doxorubicin concentrations, the drug failed to kill normal cancer cells, whereas it caused death in the KSP-siRNA-transfected cells at lower concentrations. This implies that the reduction of KSP expression mitigated the drug-resistant ability of HCC cells. These findings also further supported a correlation between drug resistance and KSP expression or signal pathways related to this protein [14,36]. Thus, the combination of siRNA and antitumor may be an efficient therapy for liver cancer treatment, which was also confirmed in recent studies [37][38][39]. Based on the results of this study, KSP is obviously the target of intense research for development of novel anti-cancer therapeutics.
Blocking KSP expression using siRNA significantly reduces KSP-mRNA and protein levels in Hep3B cells. KSP-siRNA suppresses cell proliferation and induces apoptosis through the downregulation of cyclin D1, Bcl-2, and survivin. Our data also indicated that the decrease of KSP expression made HCC cells more sensitive to anti-cancer drugs. A combination of gene therapy to reduce KSP expression and chemotherapy alleviated drug resistance in treated cells. This method could be a new targeted strategy to eradicate HCC cells in vitro.